®

Floriculture and Ornamental Biotechnology ©2012 Global Science Books

Advanced in Vivo Propagation Techniques for Specialty

Peter J.M. Knippels*

Oosterwerf 12, 2804 LZ Gouda, The Netherlands Corresponding author : * [email protected] ABSTRACT Bulbs can be propagated by natural propagation techniques like by seeds and off setts, as well as by techniques like scoring, chipping, scaling and leaf cuttings. These techniques are mostly used for in vivo propagation, less for in vitro propagation. This article concerns the first case. Specialty bulbs can successfully be propagated by scoring and chipping, either starting at the beginning of the dormant period or at the end of this period. Of various of the genera , and Ornithogalum, it is known that they can successfully be propagated by leaf cuttings. In the presented experiments the techniques scoring, chipping and leaf cuttings were applied on and Hymenocallis festalis. Scoring and chipping resulted in new newly formed bulblets with E. autumnalis. Propagation by leaf cuttings resulted only with E. autumnalis to the formation of adventitious bulblets. Crucial is the timing of propagation, which will count for all genera and species. In previous research with Lachenalia species it has been proved that the regeneration potential is highest when leaf cuttings are taken at visible bud stage.. A preliminary conclusion is that with Eucomis the regeneration potential is the highest before flowering. It is not possible to draw conclusions for Hymenocallis, because this belongs to another family than Eucomis and Lachenalia and the experiment with leaf cuttings failed with this genus. Due to the low number of used bulbs a statistical evaluation was not possible. ______

Keywords: reproduction, scoring, chipping, leaf cutting, flower , Eucomis, Hymenocallis

INTRODUCTION Scoring and chipping

General remarks The aim of this study was to determine the possibilities of propagation of specialty bulbs by scoring, portioning, with Already for decades the most used in vivo propagation as time of propagation the end of the dormant period. Also method for Lilium is scaling, as scoring is an important propagation by leaf cuttings was studied. The possibilities technique used with Hyacinthus. On a smaller scale Narcis- are determined as the propagation rate and the size of the sus and Hippeastrum are propagated by chipping. Due to formed bulbs. the lack of information, the in vivo techniques were almost Bulbous can be propagated by various “natural” not used for other bulbous crops by commercial bulb grow- propagation techniques like by seed and vegetative repro- ers. Especially in the second half of the 90’s of the 20th duction (off sets or division). Lilium bulbs can be propa- century and the first years of the 21st century applied re- gated by taking off the scales to produce bulblets on the search has been done on the in vivo propagation of specialty scales, this is known for a long period and is practiced since bulbs by chipping, scoring and partitioning. This research the years before the Second World War (Botke and Van der has led to protocols that are used on a large scale in practice Slikke 1942). In general "artificial" propagation methods by the bulb industry nowadays. The propagation techniques have been developed and improved in order to achieve a are used commercially with Galanthus and other members greater reproduction rate than can be possible by the natural of the family and Eucomis and Ornitho- methods or to obtain plants which are genetic identical to galum and other members of the Hyacinthaceae family. The the mother . The most well-known propagation tech- in vivo techniques can be applied on a large scale, with low costs, little investments and knowledge, with as a result a relatively great number of bulblets per bulb. There are Table 1 Results of the propagation of Eucomis autumnalis and Hymeno- almost no questions raised which ask for new research on callis festalis by scoring, chipping and leaf cuttings after one growing this topic. Let me give an example of this: One of the recent season: propagation rate and average circumference of bulbs formed. developments in the propagation of Zantedeschia corms is Genus and Propagation technique Propagation Average the dividing of the corms into several parts, comparable to species rate circumference partitioning with bulbs. The techniques are developed and of bulbs optimised by the growers themselves, using earlier deve- formed (cm) loped protocols, without the additional support of research. Eucomis Scoring Where in vivo propagation means little investments in autumnalis equipment and no special knowledge and education, in vitro Chipping, 4 parts 0 - propagation asks for special equipment and knowledge. Chipping, 8 parts 0 - This means that in vitro propagation is more expensive than Leaf cutting, bottom 3 < 2 in vivo. In vitro techniques are with flower bulbs used for Leaf cutting, top 0 - obtaining healthy, disease free material or for propagating Hymenocallis Scoring 9 6 material of new varieties on a large scale, as with Canna, festalis Chipping, 4 parts 8 7 Hippeastrum, Lilium, Dahlia and Zantedeschia. Chipping, 8 parts 13 4 Leaf cutting, bottom 0 - Leaf cutting, top 0 -

Received: 22 December, 2010. Accepted: 20 December, 2012. Original Research Paper Floriculture and Ornamental Biotechnology 6 (Special Issue 2), 154-157 ©2012 Global Science Books

Fig. 1 Bulblets of Hyacinthus ‘Kings of the Blues’, propagated by scoring. Fig. 2 Parts of Galtonia candicans in vermiculite with the newly formed bulblets. Fig. 3 Bulbs of Eucomis autumnalis at start of experiment, using scoring. Fig. 4 Bulb of Hymenocallis festalis at start of experiment, using chipping. Fig. 5 Hymenocallis festalis propagated by scoring, at the end of the experiment on 31 October 2010. Fig. 6 Hymenocallis festalis propagated by chipping, 8 parts at the end of the experiment. Note the numerous small bulblets on the bottom part of the bigger formed bulblets on 31 October 2010. Fig. 7 Eucomis autumnalis propagated by leaf cuttings, bottom part, at the end of the experiment on 31 October 2010. Fig. 8 Leaf cuttings of albiflos with newly formed bulblets, four months after propagation. From left to the right: bottom part leaf, middle section and top part. niques are chipping, scaling and scoring. A number of major the medium is kept moist. After about a month, the first bulbous crops such as Lilium (scaling), Hyacinthus (sco- bulblets and will have formed. Suh and Lee (1997) ring) (Fig. 1) and Narcissus (chipping) are mass reproduced described the results of leaf cutting with in this way. The techniques are for commercial use especi- 'Pearsonii'. The best results were obtained by taking the ally useful for those plants that do not reproduce easily or at bottom part of a leaf, by putting that in a potting medium of all by natural reproduction methods. Little research has equal parts peat and perlite and to keep the cuttings at a been done on the possibilities with the artificial methods for temperature of 20°C. Most recent research with Lachenalia propagation of tender, specialty bulbous plants. Most recent is done by Ndou (2000) and Niederwieser and Ndou (2002). results of experiments with representatives from this group One of the objectives in the research was the timing of the are described by Van Leeuwen and Van der Weijden (1997) propagation in relation to the regeneration potential. One of and Knippels (2000). Knippels (2000) described a chipping the conclusions was that the age of the leaf tissue affected protocol for the propagation of specialty bulbs, which can the number of formed buds. Young tissue was the best be used for deciduous or evergreen, summer growing and source of tissue. After flowering the regeneration potential winter growing bulbs. Bulbs are chipped at the beginning of decreased. Blomerus and Schreuder (2002) have described dormancy and stored, 12 weeks storage in vermiculite (Fig. the propagation of Ornithogalum thyrsoides by leaf cutting. 2), followed by planting in soil at 20-25°C, later between 15 Objectives were a.o. the influence of bottom temperature on and 20°C. the number of formed bulbs and the size of these bulbs and Van Leeuwen and Van der Weijden (1997) showed that the used medium. The largest number of bulbs were formed and can be propagated by in a peat or sand medium and a bottom temperature of 14°C. chipping, when performed in December and in April. For The biggest bulbs were produced at a bottom temperature of Eucomis bicolor there was no difference in propagation rate 24.5°C. and yield between chipping in December and storage till In this paper an experiment is described with a rep- planting in April and chipping in April and direct planting. resentative of the family Hyacinthaceae, Eucomis autum- The highest yield (a.o. number of bulbs) for Eucomis nalis, and one of the family Amaryllidaceae, Hymenocallis bicolor was obtained with chipping in December. The tests festalis, which are subjected to three propagation techniques by Knippels (2000) with Eucomis bicolor with propagation performed in vivo: scoring, chipping and leaf cuttings. at the beginning of the dormant period and storage at 20°C These techniques can also be done in vitro, but this was not for 13 weeks, gave comparable results as reported by Van within the scope of the experiment. After one growing sea- Leeuwen and Van der Weijden (1997). The results of Knip- son, after harvesting the bulbs, the propagation rate for each pels (2000) and Van Leeuwen and Van der Weijden (1997) used technique is determined. The reason to perform this are presented in Table 1. experiment was to answer the above mentioned questions and to verify the results of the experiments of Van Leeuwen Leaf cuttings en Van der Weijden (1997).

Various research has been done on the propagation of speci- MATERIALS AND METHODS alty bulbs by leaf cuttings: Krause (1980); Duncan (1988); Suh and Lee (1997); Ndou (2000); Knippels (2000, 2003); Scoring and chipping Blomerus and Schreuder (2002) and Niederwieser and Ndou (2002). Duncan (1988) described that Lachenalia In the experiments bulbs of Eucomis autumnalis (circumference species can be propagated by leaf cuttings. One or more 13 cm) and Hymenocallis festalis (circumference 18 cm) were leaves are taken from healthy, virus-free plants and, depen- used. The scoring and chipping started on 7 March 2010 with still ding on the size of the leaf, cut into several parts. The dormant bulbs. This date is about 4 to 6 weeks prior to normal leaves or parts of leaves are planted in a rooting medium. planting, so at the end of the dormancy period. Duncan mentions a medium of equal portions river sand For the propagation by scoring and chipping two bulbs of E. and vermiculite. The bottom centimetre of the leaf parts are autumnalis and one of H. festalis were used. The applied propaga- put in the medium. The whole is placed in a shady spot and tion techniques were:

155 Possibilities of in vivo propagating specialty bulbs. Peter J.M. Knippels

Table 2 Results propagation experiments Knippels (2000) 1) and Van Leeuwen and Van der Weijden (1997) 2): propagation rate and average circumference of bulbs formed. Propagation technique Propagation rate Average circumference of bulbs formed (cm) Hyacinthaceae Albuca longipes 1) Chipping 6 2 Drimiopsis maculata 1) Chipping 9 4 Eucomis bicolor 1) Chipping 14 23 Eucomis bicolor, propagation December and April 2) Chipping 11 Eucomis comosa, propagation December 2) Chipping 11 Galtionia candicans 1) Chipping 12 8 Lachenalia mathewsii 1) Chipping 3 < 2 Lachenalia unicolor 1) Chipping 1 < 2 Amaryllidaceae Crinum x powellii 1) Scoring 3 10 Haemanthus albiflos 1) Chipping 6 3 Haemanthus coccineus 1) Chipping 2 < 2 Haemanthus albiflos 1) Leaf cutting 1 to 2 Up to 4

1. scoring. Eight cuttings were made in the bottom part of the dormant period followed by a storage period in vermiculite bulbs, each cutting two cm deep (Fig. 3); or sand of 13 weeks before planting in soil?’ (Knippels 2. chipping. Bulbs divided into 4 equal parts (Fig. 4); 2000). With the performed tests with H. festalis and E. 3. chipping. Bulbs divided into 8 equal parts. autumnalis this answer cannot be given. According to Van The bulbs and the bulb parts were planted in sand with a Leeuwen and Van der Weijden (1997) at least for Eucomis a diameter with a maximum of 3 mm with the lower part of the positive answer can be given on the above raised question, bulbs and parts in the sand. The pots were placed in a shady place but can vary for the various species. at a temperature of 21-24°C. In the experiment with H. festalis a not known pheno- menon was observed. In the case of the treatment of Leaf cuttings division into 8 parts two newly formed bulbs were har- vested with numerous ‘side bulbs’ (Fig. 6). At the bottom For the propagation by leaf cuttings two bulbs of E. autumnalis part, at the basal plate of the newly formed bulbs various and one of H. festalis were planted in two liter pots on 20 March small bulblets are formed. The side bulblets were not 2010 and grown in a greenhouse. Of each plant leaves were taken formed on the basal plate of the old mother bulb. The most on 5 August 2010: of E. autumnalis 6 leaves with a length of 15- probable explanation for this behaviour is the type of re- 18 cm and of H. festalis 5 leaves of 35-40 cm length. The E. generation. Plant hormones in the basal plate and the bulb autumnalis plants were at that time in full bloom. The H. festalis parts induce adventitious bulblets, and subsequently the were non-flowering plants. Each leaf was cut into 2 parts of equal bulblets are formed on the new bulblet: a form of rejuvena- length: a bottom part and the top part. The leaf cuttings were with tion of the newly formed bulblets. Further research has to the bottom part placed in moist sand with a diameter with a be done to verify the observed phenomenon. maximum of 3 mm. The pots with the leaf cuttings were placed in a shady place at a temperature of 22-25°C. Leaf cuttings After one growing season all bulbs (end October), bulb parts and leaf sections were observed for the number and size of the In the here described experiments the leaf cuttings were newly formed bulbs. taken in the case of E. autumnalis during flowering and with H. festalis of a not . The period RESULTS AND DISCUSSION between the start of propagation by leaf cuttings and the time of the end of the experiment was 12½ weeks. During Scoring and chipping this period in the case of E. autumnalis all top leaf parts died off, as well as a part of the bottom leaf parts, most As shown in Table 2, E. autumnalis did not form any bulbs probably they dried. There is no explanation why all top after scoring and chipping. Part of the used bulbs and bulb leaf parts died off. Four bottom leaf parts could be parts stayed intact, the other parts died off. The dying off examined at the end of the experiment. In week 12 of the was not caused by a fungal infection, as no symptoms of experiment 2 small bulbs were formed (Table 2; Fig. 7). At any fungal infection were visible. The most obvious reason the bottom side of the 4 bottom leaf parts a callus like tissue that in case of E. autumnalis no adventitious bulblets were was visible. It looked like a thickening of the bottom side. formed, is that the bulbs did not receive enough temperature At first this callus like tissue is produced before adven- after chipping. titious bulbs are formed. Part of the cells in this callus tissue With H. festalis scoring and chipping resulted in the transformed into adventitious bulbs. formation of new adventitious bulbs (Fig. 5). The propaga- On internet various information (International Bulb tion rate and the average circumference of the formed bulbs Society 2004) has been published on the possibilities of were almost the same for scoring and cutting into 4 parts, as propagation of Eucomis by leaf cuttings. The results of the presented in Table 2. The bulb cut into 8 parts produced described experiment show that Eucomis can successfully more new bulbs compared with the other two techniques: be propagated by leaf cuttings. The timing of the propaga- 13 versus 8 or 9. The size of the bulbs however was with tion is not indicated. In this paper propagation of Eucomis this technique (4 cm) smaller than with the other two autumnalis started during flowering and resulted in a lim- methods (6 and 7 cm). The reason that more new bulbs are ited number of small bulbs. Based on the experiments with formed when cut into 8 parts, can most probably be ex- Lachenalia by Ndou (2000) and Niederwieser and Ndou plained by the situation that besides buds have sprouted, but (2002), the most likely conclusion can be drawn that the also adventitious bulbs are formed. With scoring and cut start of the experiment with E. autumnalis was too late. The into 4 parts only buds sprouted. limited number of formed bulbs was caused by a low propa- One of the objectives of this part of experiment was to gation potential, which seems to decrease with the flower- answer the question ‘Will chipping at the end of the dor- ing. This has to be confirmed by further experiments with mant period followed by immediate planting lead to other other Eucomis species. Propagation of bulbous plants by results compared to propagation during the first half of the leaf cuttings is in general a potential propagation technique

156 Floriculture and Ornamental Biotechnology 6 (Special Issue 2), 154-157 ©2012 Global Science Books with commercial potentials, at least for Eucomis, Lache- albiflos (Fig. 8), another member of the Amaryllidaceae nalia and Ornithogalum. Whether it offers possibilities for family, of which results are presented in Table 1. With this other genera has to be investigated. species adventitious bulblets were formed. The remark has The propagation of H. festalis by leaf cuttings resulted to be made that the leaves of Haemanthus albiflos are suc- in dying of the leaf parts soon after the experiment started. culent and won’t dry easily like with H. festalis. This is There was during the period until the leaf parts died no what most likely has happened with this species in this ex- symptoms of any fungal infection visible. periment.

CONCLUSIONS ACKNOWLEDGEMENTS

Scoring and chipping I would like to thank Paul van Leeuwen for his comments and advice. It was not possible to propagate E. autumnalis by scoring and chipping, most probably due to the point that the used REFERENCES bulbs did not receive enough temperature after chipping. The conclusion can be drawn that all three techniques can Blomerus LM, Schreuder HA (2002) Rapid propagation of Ornithogalum be applied successfully when propagated at the end of the using leaf cuttings. Acta Horticulturae 570, 293-296 dormant period with at least H. festalis. A preliminary con- Botke J, Van der Slikke CM (1942) Lelies en Leliecultuur, Rijksuitgeverij clusion is that in general the here mentioned representatives Dienst van de Nederlandsche Staatscourant, The Hague, The Netherlands, of the family of Amaryllidaceae can be propagated by 155 pp scoring and chipping, with as most suitable timing the Duncan GD (1988) The Lachenalia Handbook (1st Edn), Kirstenbosch Natio- beginning and the end of the dormant period. Experience in nal Botanic Gardens, Cape Town, South , 71 pp practice shows that with the commercial crops Narcissus, International Bulb Society (2004) Propagating Eucomis by leaf cuttings. Galanthus and Hippeastrum the best results are obtained Available online: with propagation at the beginning of the dormant period. http://www.bulbsociety.org/GALLERY_OF_THE_WORLDS_BULBS/GRA PHICS/Eucomis/Eucomis_leafcutting/Eucomis_leafcutting.html These are crops that require cold during their dormant Knippels PJM (2000) Propagation of specialty bulbs. Herbertia 55, 64-73 period. There seems to be a difference in the most appropri- Knippels PJM (2003) Poederkwasten. Succulenta 5, 215-220 (in Dutch) ate time for propagation, at the beginning or at the end of Krause J (1980) Propagation of hyacinth by leaf cuttings. Histological changes the dormant period, between Amaryllidaceae genera that in leaf cuttings of Hyacinthus orientalis. Acta Horticulturae 109, 271-277 need cold during their dormant period and those genera that Leeuwen van PJ, van der Weijden JA (1997) Propagation of specialty bulbs do not require such a period (pers. com. Van Leeuwen by chipping. Acta Horticulturae 430, 351-353 2010). Meerow AW (2001) Twin scaling of tunicate bulbs. Bulbs 2-4, 37-38 Mori G, Harai H, Imanishi H (1997) Vegetative propagation of Nerine bulbs Leaf cuttings by cross-cutting. Acta Horticulturae 430, 377-381 Ndou AM (2000) Optimization of bulblet production by leaf cuttings in Lache- nalia. PhD dissertation, Faculty of Natural and Agricultural Science, Uni- The preliminary conclusion with H. festalis is that the dying versity of Pretoria, Pretoria, , 89 pp of the leaf parts had a natural cause and that this propaga- Niederwieser JG, Ndou AM (2002) Review on adventitious bud formation in tion method is not suitable for H. festalis. More research is Lachenalia. Acta Horticulturae 570, 135-140 needed to confirm this. Knippels (2000, 2003) describes ex- Suh JK, Lee JS (1997) Leaf cutting propagation, growth and flowering of periments of propagation by leaf cuttings with Haemanthus Lachenalia. Acta Horticulturae 430, 369-376

157